High-resolution chromosome analysis of methotrexate synchronized cells from bone marrow aspirates may become an essential tool, along with the FAB cytological classification, in the subclassification of acute nonlymphocytic leukemia (ANLL) and myelodysplastic syndrome. With this technique, we routinely obtain mitoses at the 320-850 band stages and have shown that most cases analyzed have an abnormal karyotype (94 of 101 = 93% ANLL patients and 22 of 27 = 81% myelodysplastic patients) and approximately two-thirds have a specific chromosomal defect. Nearly 50% of cases of de novo ANLL and myelodysplasia were previously considered to have a normal karyotype. Our methotrexate technique for bone marrows has now been improved to yield 850-2000 bands per haploid set. This new bone marrow technique will be used to study, prospectively, some 335 consecutive new leukemic and 170 consecutive new myelodysplastic patients to correlate chromosomal data with response to the new ECOG chemotherapeutic protocol. This large study should provide critical clues to the clinical course, response to treatment, and survival patterns of individuals with specific chromosomal defects. It may also establish high resolution chromosome analysis as a critically important tool in the diagnosis, classification, prognosis, and therapeutic assessment of leukemia and myelodysplasia. The mechanisms that trigger specific chromosomal rearrangements in leukemia and cancer are unknown. Preliminary evidence from our laboratory suggests, however, that in three types of leukemia one of the breakpoints of the specific chromosomal defects found in the malignant cells can also be found in the normal blood cells of the patient as one of 17 heritable fragile sites recently defined at specific chromosome bands or subbands. These sites are often expressed when blood cells are cultured in media deprived of folate and thymidine. Standard techniques provide a low number of mitoses and percentage of clearly visualized fragile sites, making difficult their widespread use. We have now developed a highly sensitive methotrexate blood chromosome technique that appears to link the expression of heritable and spontaneous chromosomal breaks to misincorporation of uridine. This technique promises to provide evidence whether some or most leukemia patients with a specific chromosomal defect carry a fragile site in their normal cells which makes them genetically predisposed to develop a certain type of malignancy.